Insulin Signaling and Metabolic Regulation in Adipocytes

描述(申请人提供)：这一修订项目的长期目标是了解调节脂肪细胞胰岛素敏感性和代谢流量的主要分子机制。小鼠脂肪细胞葡萄糖代谢的紊乱会导致肌肉胰岛素抵抗和糖尿病，而脂肪细胞对葡萄糖的利用增加会提高全身的葡萄糖耐量。因此，识别和详细描述脂肪细胞代谢的新基因和调控基因是这一领域的基本目标。我们使用基于高通量RNAi的筛选获得了令人兴奋的初步数据，揭示了两种蛋白质作为脂肪细胞中葡萄糖和脂肪酸氧化的新的全球调节因子-辅阻遏子RIP140和蛋白激酶MAP4K4。我们的数据表明，RIP140和MAP4K4的作用都收敛于PPAR-1，这是由这些蛋白质负调控的，并且这些作用可能是高度整合的。在培养的脂肪细胞中沉默RIP140或MAP4K4：1。增强碳水化合物和脂肪酸氧化中的酶的表达，2.)增强对胰岛素敏感的葡萄糖摄取，以及3.)增强控制线粒体生物发生和氧气消耗的酶的表达。在缺乏RIP140的脂肪细胞中观察到氧化代谢显著增强。令人惊讶的是，RIP140 KO小鼠在喂食高脂肪食物时，既能抵抗肥胖，又能抵抗葡萄糖耐受。我们建议在这里确定RIP140和MAP4K4抑制和TZD调节的共同基因集，以检验PPAR？是他们监管的目标。潜在PPAR的两种机制？将对镇压进行评估：1.)PPAR之间可能存在直接相互作用？和RIP140或MAP4K4，以及，2.)PPAR的直接磷酸化？还是PPAR的监管者？例如MAP4K4的RIP140本身。对PPAR？敏感和不敏感基因的启动子区域进行芯片分析，以确定与PPAR？的复合体中是否存在RIP140或MAP4K4。在这些推动者身上。RIP140和MAP4K4对PPAR作用分子机制的实验研究？现在建议使用确认的基于TZD敏感的荧光素酶的PPAR报告？脂肪细胞中的启动子活性。质谱学将确定RIP140、PPAR？和其他MAP4K4的潜在底物上可能的磷酸化位点，然后对它们在转录调控中的功能进行分析。我们还将测试TZD对PPAR作用的分子基础。因为它与MAP4K4和RIP140功能有关。最后，新提出的研究解决了RIP140的代谢作用如何与小鼠的葡萄糖耐量有关。我们将验证这一假设，即大大增强RIP140 KO小鼠脂肪和肌肉中的脂肪酸氧化，释放脂肪酸衍生物对胰岛素信号的抑制。这些研究将揭示这些新的脂肪细胞代谢和功能的全球调节因子的潜在作用机制。

DESCRIPTION (provided by applicant): The long-range goal of this revised project is to understand major molecular mechanisms that regulate insulin-sensitivity and metabolic flux in adipocytes. Disruptions in adipocyte glucose metabolism in mice can cause muscle insulin resistance and diabetes, while augmenting glucose utilization in adipocytes enhances whole body glucose tolerance. Thus, the identification and detailed characterization of novel genes and regulators of adipocyte metabolism are fundamental objectives in this field. Exciting preliminary data we obtained using high-throughput RNAi-based screens have revealed two proteins as novel global regulators of glucose and fatty acid oxidation in adipocytes - the corepressor RIP140 and the protein kinase MAP4K4. Our data suggest the hypothesis that the actions of both RIP140 and MAP4K4 converge at PPAR-?, which is negatively regulated by these proteins, and these actions may be highly integrated. Silencing either RIP140 or MAP4K4 in cultured adipocytes: 1.) enhances expression of enzymes in carbohydrate and fatty acid oxidation, 2.) enhances insulin-sensitive glucose uptake, and 3.) enhances expression of enzymes that control mitochondrial biogenesis and oxygen consumption. Markedly increased oxidative metabolism is observed in adipocytes lacking RIP140. Strikingly, RIP140 KO mice are resistant both to obesity and to glucose intolerance when fed a high fat diet. We propose here to identify the set of genes that is common to RIP140 and MAP4K4 repression and TZD regulation to test the hypothesis that PPAR? is a target of their regulation. Two mechanisms of potential PPAR? repression will be evaluated: 1.) possible direct interactions between PPAR? and RIP140 or MAP4K4, and, 2.) direct phosphorylation of PPAR? or regulators of PPAR? such as RIP140 itself by MAP4K4. ChIP analysis of promoter regions of PPAR?-sensitive and insensitive genes will be performed to determine whether RIP140 or MAP4K4 are present in complexes with PPAR? at these promoters. Experiments to probe molecular mechanisms of RIP140 and MAP4K4 actions on PPAR? are now proposed, using a confirmed TZD-sensitive luciferase-based reporter of PPAR? promoter activity in adipocytes. Mass spectrometry will identify possible phosphorylation sites on RIP140, PPAR?, and other potential substrates of MAP4K4, followed by their functional analysis in transcriptional regulation. We shall also test the molecular basis of TZD action on PPAR? as it relates to MAP4K4 and RIP140 functions. Finally, newly proposed studies address how the metabolic actions of RIP140 are linked to glucose tolerance in mice. We shall test the hypothesis that greatly enhanced fatty acid oxidation in fat and perhaps muscle in RIP140 KO mice releases the inhibition by fatty acid derivatives on insulin signaling. These studies will reveal underlying mechanisms of action of these novel global regulators of adipocyte metabolism and function.